The invention relates to the identification of isolated, full-length human MEKK1 nucleic acid molecules encoding MEKK1 proteins, and methods of using the nucleic acid molecules and proteins.
Controlling the state of phosphorylation is an important mechanism by which signaling molecules regulate the activity of other proteins. One common theme in such molecular signaling is the so-called kinase cascade, in which a linear series of kinases is activated by phosphorylation by upstream kinases. The mitogen-activated protein kinase (MAPK) pathway is one such example of a kinase cascade.
MAPKs are activated and phosphorylated by MAPK kinases (MKKs). The MKKs, in turn, are activated and phosphorylated by serine/threonine kinases (MKKKs), which themselves may be activated and phosphorylated by MKKK kinases (MKKKKs). There are currently over ten different groups of kinases covering more than twenty-two different genes that act upstream of one another and regulate the MKKs. One family, the MAPK/ERK kinase kinases (MEKKs) directly activate and phosphorylate specific MKKs. (Schlesinger et al., Front Biosci. 3:d1181-1186 (1998).)
MEKKs are activated by a number of diverse extracellular stimuli, indicating that not only can these molecules affect a wide variety of downstream activity, but they can also react to a diverse array of extracellular stimuli. For example, both EGF receptor stimulation and TNF a lead to an increase in MEKK1 activity. (Lange-Carter et al., Science 265:1458-1461 (1994); Winston et al., Proc. Natl. Acad. Sci., 92:1614-1618 (1995); Ishizuka et al., J. Biol. Chem. 271:12762-12766 (1996); Kaga et al., J. Immunol. 160:4182-4189 (1998).) MEKK1 is also activated in response to DNA damaging stresses such as UV irradiation, etoposide, cisplatin, and mitomycin C. (Widmann et al., Mol Cell. Biol. 18:2416-2429 (1998); Cardone et al., Cell 90:315-323 (1997).)
To date, six different MEKK genes have been at least partially cloned in mammalian cells. (Lange-Carter et al., Science 260:315-319 (1993); Blank et al., J. Biol. Chem. 271:5361-5368 (1996); Gerwins et al., J. Biol. Chem. 272:8288-8295 (1997); Wang et al., J. Biol. Chem 271:31607-31611 (1996); Wang et al., Biochem. Biophys. Res. Commun. 253:33-37 (1998).) There are a number of functional motifs found within the N-terminal regulatory domains of, at least, MEKK1 and MEKK4. Both molecules contain putative pleckstrin homology domains. Pleckstrin domains associate with polyphosphoinositides and mediate localization to specific regions of the plasma membrane. MEKK1 and MEKK4 also contain proline rich regions at the N-terminus, which may be of functional significance. Proline rich regions have been shown to be important for binding proteins that contain Src homology 3 (SH3) domains. Moreover, with regard to MEKK1, 14-3-3 proteins bind at the N-terminal regulatory domain. Although 14-3-3 association does not appear to dramatically affect MEKK activity, 14-3-3 proteins are important for MEKK regulation by mediating interactions with other regulatory proteins and for controlling subcellular localization of these kinases.
MEKK1 is important in regulating cell survival and apoptosis. MEKK1 is a substrate for caspases, a family of proteases required for apoptosis. The apoptotic signaling appears to be dependent on cleavage of MEKK1 and subsequent caspase activation, as cleavage resistant mutants do not induce apoptosis. Thus, MEKK1 plays a critical role in regulating cell survival and death, acting as a molecular switch when cleaved by caspases. Cleavage by a caspase changes MEKK1 from a survival promoting kinase to an effector of cell death.
MEKK1 activates both the Activator Protein-1 (AP-1) stress response pathway and the NFxcexaB pathway. The transcription factor AP-1 is a critical regulator of T-cell activation, cytokine production, including IL-2, IL-3, and GM-CSF, and the production of metalloproteinases. (Gottschalf et al., J. Exp. Med. 178:1681 (1993); Want et al., J. Mol. Cell. Biol. 14:11153 (1994); Rao, Immunol. Today 15:274 (1994); Angel et al., Biochem. Biophys. Acta. 1072:129 (1991).) With regard to cytokine regulation, AP-1 mediates positive transactivation independently or in association with NF-AT (Nolan, Cell 77:795 (1994)). AP-1 activity is induced by many stimuli, including the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), growth factors, cytokines, T-cell activators, neurotransmitters, and UV irradiation. AP-1 is composed of dimers of different members of the Fos and Jun family of proteins. AP-1 activity is regulated at the level of both C-Jun and C-Fos transcription and by post-translational modification of their protein products by phosphorylation and dephosphorylation.
Moreover, independent of its MAPK activity, MEKK1 also plays another role in regulating transcription factor NFxcexaB, which is a dimer maintained in the cytoplasm via an inhibitory regulatory subunit, IxcexaB. (Lee et al., Cell 88:213 (1997).) Upon stimulation with specific cytokines or environmental stresses, IxcexaB is phosphorylated by IxcexaB kinase which induces proteolytic degradation of IxcexaB, thereby releasing NFxcexaB to translocate to the nucleus effecting changes in transcription. (PCT Publication No. WO 97/35014.) Over expression of MEKK1 activates NFxcexaB.
The two transcription factors, NFxcexaB and AP1, have been shown to regulate the production of many proinflammatory cytokines and related proteins that are elevated in immunoinflammatory diseases. These transcription factors regulate IL-1, IL-2, TNF xcex1, IL-6, and IL-8 levels in a variety of cell types. NFxcexaB and other related transcription factor complexes are involved in the rapid induction of genes whose products function in protective and proliferative responses upon exposure of cells to external stimuli. Similarly, AP-1 has a significant role in the regulation of IL-2 transcription during T-cell activation. Thus, the role of NFxcexaB and AP-1 is to act as a transducer of certain stimuli that lead to immune, inflammatory, and acute phase responses and, when overactivated, can lead to a disease state (Suto et al., Current Pharm. Design 3:515-528 (1997). No known antiinflammatory or autoimmune drugs have been specifically developed clinically as inhibitors of NFxcexaB or AP-1. Therefore, a critical need exists for new therapies to treat immunoinflammatory and autoimmune disorders.
Prior to the instant invention, cloning efforts to obtain the full-length human MEKK1 gene have been unsuccessful for several reasons. Earlier efforts by others failed to identify the full-length cDNA. In some instances, the libraries that were screened were 5xe2x80x2-stressed and would not have expressed a full-length coding region. Specifically, the 5xe2x80x2 region of the gene proved difficult to clone for reasons that were, until now, unclear. Other attempts using reverse PCR methodology were also unsuccessful.
Nevertheless, in order to completely understand MEKK1 biology and to develop potential regulators or modulators of MEKK1, the full-length MEKK1 nucleic acid molecule had to be cloned and isolated. There is a need, 9 therefore, to identify and clone the full-length MEKK1 nucleic acid to further understand and exploit the role of MEKK1.
The present invention is based, in part, on the discovery of a novel fill-length human gene referred to herein as xe2x80x9cMEKK1xe2x80x9d. The polynucleotide sequence of a cDNA encoding a MEKK1 polypeptide is shown in SEQ ID NO:1, and the amino acid sequence of a MEKK1 polypeptide is shoen in SEQ ID NO:2. In addition, the polynucleotide sequence of the coding region is from nucleotide 7 to nucleotide 4545 of SEQ ID NO:1.
Accordingly, in a first aspect, the invention features a full-length nucleic acid molecule which encodes a MEKK1 protein or polypeptide, e.g., a biologically active portion of the MEKK1 protein. In a preferred embodiment the isolated nucleic acid molecule encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. In other embodiments, the invention provides isolated MEKK1 nucleic acid molecules having the polynucleotide sequence shown in SEQ ID NO:1, or nucleotide 7 to nucleotide 4545 of SEQ ID NO:1, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number PTA-1836. In still other embodiments, the invention provides nucleic acid molecules that are substantially identical (e.g., naturally occurring allelic variants) to the nucleotide sequence shown in SEQ ID NO:1, or nucleotide 7 to nucleotide 4545 of SEQ ID NO:1, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number PTA-1836. In other embodiments, the invention provides a nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the polynucleotide sequence of SEQ ID NO:1 or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number PTA-1836, wherein the nucleic acid encodes a full-length MEKK1 protein or an active fragment thereof.
In a related embodiment of the first aspect, the invention further provides nucleic acid constructs which include a MEKK1 nucleic acid molecule described herein. In certain embodiments, the nucleic acid molecules of the invention are operatively linked to native or heterologous regulatory sequences. Also included are vectors and host cells containing the MEKK1 nucleic acid molecules of the invention, e.g., vectors and host cells suitable for producing MEKK1 nucleic acid molecules and polypeptides.
In another related embodiment of the first aspect, the invention provides nucleic acid fragments suitable as primers or hybridization probes for the detection of MEKK1-encoding nucleic acids.
In still another related embodiment of the first aspect, isolated nucleic acid molecules that are antisense to a MEKK1 encoding nucleic acid molecule are provided.
In a second aspect, the invention features MEKK1 polypeptides, and biologically active or antigenic fragments thereof, that are useful, e.g., as reagents or targets, in assays applicable to treatment and diagnosis of MEKK1 mediated or related disorders. In another embodiment, the invention provides MEKK1 polypeptides having a MEKK1 activity. Preferred polypeptides are MEKK1 proteins including at least one processed/cleaved domain, e.g., amino acid residues 876-1512 of SEQ ID NO:2, and/or one kinase or catalytic domain, e.g., amino acid residues 1191-1512 of SEQ ID NO:2, and, preferably, having a MEKK1 activity, e.g., a MEKK1 activity as described herein.
In another embodiment of the second aspect, the invention provides MEKK1 polypeptides, e.g., a MEKK1 polypeptide having the amino acid sequence shown in SEQ ID NO:2; the amino acid sequence encoded by the cDNA insert of the plasmid deposited with ATCC Accession Number PTA-1836; an amino acid sequence that is substantially identical to the amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence encoded by a nucleic acid molecule having a polynucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the polynucleotide sequence of SEQ ID NO:1 or nucleotide 7 to nucleotide 4545 of SEQ ID NO:1, or the sequence of the DNA insert of the plasmid deposited with ATCC Accession Number PTA-1836, wherein the nucleic acid encodes a full length MEKK1 protein or an active fragment thereof.
In a related embodiment of the second aspect, the invention further provides nucleic acid constructs that include a MEKK1 nucleic acid molecule described herein.
In a related embodiment of the second aspect, the invention provides MEKK1 polypeptides or fragments operatively linked to non-MEKK1 polypeptides to form fusion proteins.
In a third aspect, the invention features antibodies and antigen-binding fragments thereof, that react with, or more preferably specifically bind MEKK1 polypeptides.
In a fourth aspect, the invention provides methods of screening for compounds that modulate the expression or activity of the MEKK1 polypeptides or nucleic acids.
In a fifth aspect, the invention provides a process for modulating MEKK1 polypeptide or nucleic acid expression or activity, e.g., using the aforementioned screened compounds. In certain embodiments, the methods involve treatment of conditions related to aberrant activity or expression of the MEKK1 polypeptides or nucleic acids, such as conditions involving aberrant or deficient cellular adhesion, proliferation or differentiation.
The invention also provides assays for determining the activity of, or the presence or absence of, MEKK1 polypeptides or nucleic acid molecules in a biological sample, including for disease diagnosis.
In a sixth aspect, the invention provides assays for determining the presence or absence of a genetic alteration in a MEKK1 polypeptide or nucleic acid molecule, including for disease diagnosis.
It is to be understood from the foregoing general description that the following detailed description is exemplary and explanatory, and intended to provide further explanation of the invention claimed.